The present study describes two novel compound heterozygous mutations, c. 410C>T(p. T137M) (T137M) on the maternal and g. 4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276-280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-P-i cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137] hNaPi-IIc, or EGFP[Stop446] hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent P-33 uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137] hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137] hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g. 4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of Na-22 and P-33 uptake was increased to 7.1 +/- 3.65 for EGFP[M137] hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137] hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.
